Choleragen activates adenylate cyclase by catalyzing the ADP-ribosylation of a guanyl nucleotide-binding regulatory protein (G/F). Choleragen and certain hormones are believed to activate adenylate cyclase, at least in part, by decreasing the affinity of G/F for guanyl nucleotides. Consistent with this hypothesis were the observations that both choleragen and certain hormnes stimulate the release of a pool of GDP from turkey erythrocyte membranes. It has not been demonstrated that this pool of GDP was indeed released from G/F . We have now obtained evidence that the protein which releases GDP upon incubation with choleragen is identical to G/F. After solubilization, the protein which releases GDP upon choleragen treatment cochromatographed with G/F activity. Moreover, the only protein which chromatographed as a protein of this size and which was ADP-ribosylted by choleragen was G/F. These data provide further evidence that choleragen treatment decreases the affinity of G/F for GDP. The kinetics of toxin-stimulated release of GDP from membranes was also examined.